Pbx1 is a proto-oncogene that was originally discovered at the site of chromosomal translocations in pediatric acute leukemia. It codes for a TALE class homeodomain transcription factor, which is a component of hetero-oligomeric protein complexes that regulate developmental gene expression. Using genetic approaches, we are investigating the contributions of Pbx1, and the related Pbx2 and Pbx3 proteins, to development and tissue homeostasis. Our studies are beginning to^define molecular pathways that are critically dependent on Pbx functions, most prominent of which are pathways regulated by Hox and orphan homeodomain proteins. In addition, our studies have highlighted cellular populations and processes that are critically dependent on Pbx function. A consistent observation in multiple organ systems is the reduction in proliferating progenitors, however it is unclear whether this is a primary deficiency in progenitor proliferation or a defect in the production of progenitors from tissue-specific stem cells. The resolution of this issue requires an ability to analyze the fates of single phenotypically and functionally defined cells in vivo and in vitro. The hematopoietic compartment is particularly well suited to such analyses given the defined lineal relationships of stem cells and progenitors, and our ability to prospectively isolate distinct progenitor populations. The studies in this application will address the hypothesis that Pbx proteins coordinate cellular processes that underlie the decision of hematopoietic progenitors to self-renew or differentiate. Through four specific aims we will: determine Pbx contributions to HSC self-renewal and multi-lineage differentiation; characterize specific defects in committed progenitors, their proliferation potential, and generation from stem cells; define transcriptional and signaling pathways affected by the absence of Pbx proteins in stem and progenitor cells; and characterize the functional cooperation among Pbx isoforms in hematopoiesis. Given that Pbx1 is a proto-oncogene that is targeted by chromosomal translocations in B cell progenitor and myeloid leukemias, our studies will be particularly relevant for defining Pbx roles in stages of hematopoiesis that serve as the setting for hematologic malignancies initiated by malfunction of Pbx/Hox transcriptional regulatory pathways.